The identification of over 80 autoimmune diseases in humans has led to the realization that a breakdown in self-tolerance accounts for many of the most devastating chronic diseases affecting human health worldwide. Over the past 10 years, it has become apparent that this breakdown in self-tolerance is in large part due to a loss of immune regulation, most prominently a consequence of defective regulatory T cells (Treg) and their inability to control pathogenic immunity. This conclusion has been reached based on numerous studies in animal models of autoimmunity, direct evidence in humans and genetic studies highlight the critical role of this T cell subset. These master regulators of immunity arise both in the thymus (called natural Tregs, nTregs) and in the peripheral immune system (called adaptive Tregs, aTregs) as a consequence of exposure to self-antigens. A loss of Tregs results in a fatal autoimmune disease (termed IPEX in humans), and small changes in Treg numbers or function accompany the onset of autoimmune diseases. In fact, recent data suggest that these regulatory T cells may be altered during disease progression and, in some cases, become so unstable such that they may themselves participate in destructive autoimmunity. Data from patients with a variety of autoimmune diseases has increasingly suggested that Tregs are defective. Several of the susceptibility genes identified in genome studies have shown that several genes that increase the risk for autoimmunity encode variant proteins that control Treg develop and function. However, the basis for these Treg defects and the relative contributions of nTregs versus aTregs in autoimmunity remain controversial. We hypothesize that nTregs are intimately involved in overall immune homeostasis, while aTregs that develop as a consequence of inflammation are critical for controlling local immunity and as such are most susceptible for destabilization. Recent studies in our group using gene expression arrays has identified a unique signature that distinguishes nTregs from aTregs, thus enabling, perhaps for the first time, an ability to distinguish and study these subsets. The goal of this application is to test this hypothesis in mouse models of experimental autoimmune encephalomyelitis, to define the relative importance and functional stability of Tregs in different phases of disease and to determine the relative effectiveness of nTreg versus aTregs in controlling disease activity. To test this hypothesis, we propose the following specific aims: 1. To characterize specific attributes and contributions of nTregs and aTregs in protection from autoimmune disease; and 2. To examine the stability of Tregs during autoimmune disease pathogenesis. We anticipate that the results of these studies will enable a more robust understanding of Treg function in humans with autoimmunity, provide new tools for tracking Treg function during disease progression and facilitate our ability to exploit Tregs for therapeutic usage.